Monarda fistulosa (sometimes also known as horsemint, Oswego tea, bergamot or bee balm) is a member of the Lamiaceae (Mint) family. It is a native mid-western prairie perennial plant species which propagates via slender rhizomes and via seed. It can be vegetatively propagated via rooted cuttings to generate clonal lines from a single plant. When the term “Monarda” is used herein it is understood that it includes plants known by these names, as well, provided the plant meets the characteristics of the Monarda genus.
Monarda fistulosa is common to roadsides, meadows, native prairie conservation sites and woodland borders, and was highly prized by native Americans and later pioneer settlers for its medicinal properties.
Monarda fistulosa was introduced into the Materia Medica in the 1850s due to its medicinal properties. Leaves, stems and flowers are all used medicinally with greater than 17 bioactive molecules identified, including thymol and carvacrol. Author Michael Wood (in his book Herbal Wisdom) describes this species by one of its indigenous names “sweet leaf” as one of the most valuable plants in his herbal cupboard.
Monarda fistulosa is very similar to oregano (Origanum vulgare) and thyme (Thymus vulgaris) in its ability to accumulate thymol and carvacrol in its leaves and other plant parts. Monarda fistulosa has been known to produce thymoquinone since at least as early as 1907 (Wakeman, Pigments of Flowering Plants, Transactions of the Wisconsin Academy of Science, Arts and Letters, Volume 19, pp 785-795 (1907)).
What was not previously known was that during senescence of leaves or flowers, carvacrol and thymol are converted to thymoquinone (TQ) which therefore accumulates in dead or dying tissue. Thymoquinone does not accumulate to any degree in fresh tissue. Genetic variation exists for the accumulation of (conversion from carvacrol and thymol) thymoquinone in dead, dying or post-harvest tissue just as it does for the accumulation of carvacrol and thymol in healthy fresh tissue. Accordingly, any person skilled in the art of plant breeding can select plants that accumulate high levels of carvacrol or thymol (or thymoquinone from senescent tissue) from a segregating population of plants in order to optimize accumulation levels.
Several hundred seed can be planted and grown under a uniform controlled or field environment and once the plants are in full vegetative stage, leaf tissue can be taken for phytochemical analysis of carvacrol and thymol. Day length needs to be greater 12 hours with light intensity similar to full sunlight as accumulation is day length and light intensity driven. Individual plants within a population (clonal lines) that accumulate extreme levels of carvacrol and thymol (>3% on a dry matter basis) can be selected and propagated (scaled) either vegetatively or by seed similar to other crops. Populations of plants of Monarda fistulosa can be improved for accumulation of carvacrol and thymol using a well-known breeding technique of mass selection, resulting in seeded varieties that hyper-accumulate these molecules.
Any person skilled in the art of agronomy and field crop production can grow a crop of improved Monarda fistulosa, selected for higher accumulation of carvacrol or thymol in large acreages in order to generate biomass for harvest and extraction using well known and frequently practiced agronomic methods.
An early example of a method for commercial production of thymol from a closely related species (Monarda punctata) was described in 1916 in USDA Bulletin 372 by S. C. Hood.
Planting density can be optimized to (a) reduce weed pressure; and (b) optimize biomass accumulation per unit area. The United States Department of Agriculture Natural Resources Conservation Division recommends a planting density for Monarda fistulosa of 3.6 lbs per acre. Further, in a study published in 1916, it was recommended that the planting density be such that the usual cultivation methodologies result in plants that shade the ground and thus prevent the growth of weeds (1916 in USDA Bulletin 372, ibid.).
Those skilled in the art of steam distillation can generate essential oil of Monarda fistulosa which is high in carvacrol or thymol from freshly harvested biomass; or with increasing levels of thymoquinone from senescing or senescent biomass. Steam distillation followed by subsequent decanting of the oil is a frequently practiced process for generating essential oil. Carvacrol and thymol oils are generated from oregano using this method.
Unique, hyper-accumulating clonal lines for carvacrol and/or thymol of Monarda fistulosa represent optimum assemblages of alleles derived through breeding and selection.
Wild Bergamot (Monarda fistulosa L.) is a native Iowa wildflower species in the mint (Lamiaceae) family, and is a potential source of thymoquinone. Thymoquinone is a molecule of interest for use as a functional molecule for dandruff control in shampoos and other personal care products. Thymoquinone is known for its anti-inflammatory, antimicrobial, antioxidant and immunomodulatory effects and is used for treating skin rashes, insect bites, and sun burns. It has also been shown to have significant anti-cancer activity carvacrol and its geometrical isomer thymol can be oxidized to thymoquinone, which has a potential commercial value considerably higher than its precursor's thymol and carvacrol (FIG. 1). The essential oils derived from plant species such as oregano are dominated by carvacrol and thymol while essential oils from wild bergamot and Nigella sativa are dominated by thymoquinone which occurs less frequently in nature.
Development of proprietary Monarda clonal lines with high levels of thymoquinone (thymoquinone levels ≧2.5%) combined with high biomass will result in an economically viable source of thymoquinone.
In addition to inherent genetic variation, the accumulation of these molecules is influenced by physiological maturity of the plants and the environmental conditions in which they are grown. The composition and quantity of the essential oils in several Lamiaceae species are known to change with physiological maturity and environmental conditions such as day length and light intensity. Little research has been done in understanding the variation for thymoquinone content among Monarda collections; nor time of harvest for maximizing thymoquinone content in the essential oils. The highest level of thymoquinone reported in a Monarda collection was 0.36% on a dry matter basis in flowers and 0.26% in leaves and stems. The amounts of carvacrol, thymol and other phenolic compounds differ relative to physiological maturity of the plants and flowers and therefore, distillation of flower heads harvested at different maturity stage may result in oil having different profiles.
Another study completed in 1993 looked at Monarda as a source of certain oils, specifically, geraniol, linalool, thymol and carvacrol. This study also presented the idea that plants of a given species but grown and maintained in different geographical regions may yield different oils. (Mazza, G., F. A. Kiehn, and H. H. Marshall, 1993, Monarda: A source of geraniol, linalool, thymol and carvacrol-rich essential oils, p. 628-631. In: J. Janick and J. E. Simon (eds.), New crops, Wiley, New York.)
It is well known that planting density can affect the oil yield and composition in plants. For example, in dill (Anethum graveolens L.), increasing plant density increased the amount of phellandrene, α-pinene and dill ether (Callan, N. W. et al. Industrial Crops and Products 25 (2007) 282-287). The oil yield of parsley (Petroselinum crispum) was found to be increased when the plant density of either flat-leafed or curly-leafed parsley was increased (Petropoulos, S. A. et al. Scientia Horticulturae 115 (2008) 393-397). Similarly, increasing plant density increased the essential oil production in German chamomile (Matricaria chamomilla L.) (Pirzad, A. et al. Australian Journal of Agricultural Engineering 2(5) (2011) 120-126).
Given that agronomic methods for efficiently growing Monarda fistulosa on a commercial scale and collecting the essential oils containing carvacrol and/or thymol are well within the skill in the art, what is needed are varieties of Monarda fistulosa that hyper-accumulate carvacrol and or thymol and a method of converting such compounds efficiently into thymoquinone and/or thymohydroquinone.